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Abstract

Background

Urban particulate matter (PM) has been epidemiologically correlated with multiple
cardiopulmonary morbidities and mortalities, in sensitive populations. Children exposed
to PM are more likely to develop respiratory infections and asthma. Although PM originates
from natural and anthropogenic sources, vehicle exhaust rich in polycyclic aromatic
hydrocarbons (PAH) can be a dominant contributor to the PM2.5 and PM0.1 fractions and has been implicated in the generation of reactive oxygen species (ROS).

Objectives

Current studies of ambient PM are confounded by the variable nature of PM, so we utilized
a previously characterized ethylene-combusted premixed flame particles (PFP) with
consistent and reproducible physiochemical properties and 1) measured the oxidative
potential of PFP compared to ambient PM, 2) determined the ability of PFPs to generate
oxidative stress and activate the transcription factor using in vitro and ex vivo models, and 3) we correlated these responses with antioxidant enzyme expression in vivo.

Methods

We compared oxidative stress response (HMOX1) and antioxidant enzyme (SOD1, SOD2,
CAT, and PRDX6) expression in vivo by performing a time-course study in 7-day old neonatal and young adult rats exposed
to a single 6-hour exposure to 22.4 μg/m3 PFPs.

Results

We showed that PFP is a potent ROS generator that induces oxidative stress and activates
Nrf2. Induction of the oxidative stress responsive enzyme HMOX1 in vitro was mediated through Nrf2 activation and was variably upregulated in both ages. Furthermore,
antioxidant enzyme expression had age and lung compartment variations post exposure.
Of particular interest was SOD1, which had mRNA and protein upregulation in adult
parenchyma, but lacked a similar response in neonates.

Conclusions

We conclude that PFPs are effective ROS generators, comparable to urban ambient PM2.5, that induce oxidative stress in neonatal and adult rat lungs. PFPs upregulate a select
set of antioxidant enzymes in young adult animals, that are unaffected in neonates.
We conclude that the inability of neonatal animals to upregulate the antioxidant response
may, in part, explain enhanced their susceptibility to ultrafine particles, such as
PFP.

Keywords:

Introduction

Urban particulate matter (PM) exposure has been epidemiologically correlated with
multiple cardiopulmonary morbidities and mortalities, especially in susceptible populations
[1-4]. Of special concern are the smaller fractions of PM: fine PM2.5 particles with an aerodynamic diameter (AD) of less than 2.5 μm, and ultrafine PM0.1 particles with AD of less than 0.1 μm. These fractions can penetrate deep into the
bronchiolar and alveolar regions of the lung [5]. Although particulate pollution originates from both natural and anthropogenic sources,
vehicle exhaust can be a dominant contributor of the PM2.5 and PM0.1 fractions [6]. Vehicle exhaust from combustion of gasoline, diesel and other petroleum fuels contains
carbonaceous particles with fused and free polycyclic aromatic hydrocarbons (PAHs).
It has been estimated that vehicle exhaust contributes to over 50% of urban PM2.5 mass [7].

Children are a susceptible population to inhaled PM. Several physiological factors
play a role in enhanced susceptibility. Compared to adults, children are more aerobically
active outdoors, have a larger body surface area-to-volume ratio, higher metabolic
rate, and have increased minute ventilation and oxygen consumption [8]. Moreover, the lung continues to mature, develop and grow postnatally. The developing
lung undergoes continuous alveolarization, cellular maturation and differentiation
that occurs up to the first 8 years of a child’s life [9,10]. This may be disrupted by exposure to air pollution during these critical years.
Epidemiologic studies have shown that children who live near roadways with high levels
of vehicle traffic are more predisposed to both the development and exacerbation of
asthma, have a higher incidence of pneumonia and bronchitis and are more likely to
miss school as a result [11]. PM0.1 exposure has also been linked to diminished lung development and reduced lung function
in children [12].

Reactive oxygen species (ROS) generated from PM have been implicated in the generation
of cellular oxidative stress in both in vivo and in vitro models [13,14]. Oxidative stress occurs upon a loss of cellular homeostasis, where ROS generation
overwhelms antioxidant defenses. This can directly result in cytotoxicity [15] and the activation of specific transcriptional pathways, such as Nrf2, that are responsible
for upregulating phase II antioxidant gene expression [16]. The lung is rich in non-enzymatic antioxidants, such as reduced glutathione, ascorbic
acid (Vitamin C), α-tocopherol (Vitamin E), lycopene, and β-carotene [17]. Further, systems of persistent and inducible antioxidant enzymes are uniquely equipped
to reduce intracellular ROS [18,19], restoring cellular homeostasis.

Many toxicological studies use field-collected ambient PM as the exposure regimen.
Results from these studies are confounded by the variable nature of ambient PM. The
composition of ambient PM is dependent on the time of day, season, weather and location.
This complicates the systematic attribution of health effects to a single component
or group of components and makes exact reproduction of such studies challenging. To
address this issue, we have developed and characterized a system to generate premixed
flame particle (PFP) [20] that are rich in PAHs, especially naphthalenes, to simulate the effects of inhaling
vehicle exhaust. We have previously shown that animals exposed to PFPs have increased
cellular toxicity. Further, following exposure there are age-dependent and lung compartmental
changes in levels of non-enzymatic (GSH, GSSG) and enzymatic (GCL, GSR, GPX, GSTs)
antioxidants [21-23].

Building upon our previous work, the current study was designed to address three goals:
(1) to determine whether PFPs generate ROS, (2) to examine whether changes in enzymatic
antioxidant expression in vivo are correlated with ROS generation, and (3), to examine age-specific antioxidant
expression in response to PFP exposure. We hypothesized that PFPs generate ROS, which
causes lung oxidative stress, and that the inability of neonates to upregulate antioxidant
enzymes in response to elevated oxidative stress results in enhanced epithelial susceptibility.
To test our hypothesis, we divided the current study into two parts. First, we analyzed
the oxidative potential of PFPs by measuring ROS generation in a surrogate lung fluid
(SLF), and reinforced these results using in vitro and ex vivo treatment models. In the second part, we performed a time-course study where 7-day
old neonatal pups and 8-week old young adult rats were whole-body exposed to an acute
6-hour exposure of PFPs, and we then compared antioxidant enzyme expression that can
functionally reduce the measured ROS.

Results

PFPs generate reactive oxygen species

As shown in Figure 1A, molecular oxygen is a viable electron acceptor that generates ROS (e.g. superoxide,
hydrogen peroxide and hydroxyl radicals), both through cellular respiration and production
by PM [24,25]. To define the metals concentration in PFP, which can function as a source of ROS
in addition to PAHS, we quantified 8 transition metals in collected PFP. The concentrations
of transition metals in PFP were all below the limit of quantification, with the exception
of zinc. In contrast, metal concentrations are much higher in a typical ambient PM1.8 sample from Fresno, California (Table 1). PAH concentrations were also compared using historical data [21,26]. As shown in Table 2, PAH species were present in sub-nanogram per cubic meter concentrations in both
PFP and ambient PM1.8, but distribution of PAHs differed between the two particle types. Higher concentrations
of benz(a)anthracene and pyrene species were detected in PFP, but substituted fluoranthene
and perylene species were undetectable, as compared with ambient PM1.8. To quantify the oxidative potential of PFPs, we measured dithiothreitol (DTT) consumption
as well as the generation of hydrogen peroxide (HOOH) and hydroxyl radical (·OH) in
a surrogate lung fluid. As a comparison, we also measured the oxidative potential
of the same Fresno PM1.8 sample listed in Table 1. Since the oxidative potential of ambient PM differs from sample to sample, this
Fresno sample is simply an example and not an average ambient response. Figure 1B illustrates that PFPs can generate ROS: they can both oxidize DTT and produce HOOH.
PFPs do not generate ·OH; this is consistent with their very low transition metal
concentrations, since metals are thought to be the dominant pathway for converting
HOOH into ·OH. In contrast, the typical Fresno PM sample does produce ·OH (Figure 1B); likely because of its high levels of soluble iron and copper (Table 1). However, PFPs and Fresno PM have a similar response in the DTT and HOOH assays,
indicating that PFPs can produce similar oxidative potential as ambient PM.

PFP induces cellular oxidative stress

Next, we visualized the production of oxidative stress in the conducting airway epithelium
in situ by instilling PFPs and CellROX Deep Red fluorescent oxidative detector in an ex-vivo model that preserves the 3-dimensional architecture and normal cell populations of
the conduction airways. CellROX, a cell-permeable probe that fluoresces upon oxidation
by ROS was used as a marker of oxidative stress. Figure 2 shows a time-course experiment comparing 1 hour of treatment of either PFP or PBS
(vehicle control) instilled directly into rat bronchi in a proximal to distal direction.
CellROX staining, indicative of oxidative stress, was not observed in either PBS or
PFP instilled airways immediately after 1 hour of treatment (Figure 2A, D). Bronchiolar epithelial oxidant stress was absent in PBS controls. However,
we observed mild parenchymal staining, possibly due to trauma during the dissection
preparation process or from cellular instability during continuous imaging. Nevertheless,
2 hours following the 1 hour PFP treatment, CellROX fluorescence was seen in the cells
linings the lumen of the treated bronchi (Figure 2E). This progressed over time in a proximal to distal manner (Figure 2F). This can be seen more clearly in the time-course videos, which are presented in
the Supplement: PBS control (Additional file 1: Video S1), PFP treated (Additional file 2: Video S2).

Figure 2.Ex vivo oxidative stress detection. Rat right cranial lobes were dissected to expose the main axial airway and airways
were treated with either PFP or PBS. Lungs were incubated with CellROX, a fluorescent
dye that indicates oxidative stress, and washed with Live Cell Imaging solution after
1 hour of exposure and imaged continuously. Representative pictures from lungs taken
directly (A, D), 2 hours (B, and E) and 4 hours (C and F) after a 1 hour treatment of either PBS (A-C) or PFP (D-F). Substantially more CellROX fluorescence could be observed in the airway lumen of
the PFP-treated lung compared to PBS controls in a time dependent and proximal (PROX)
to distal (DIST) manner. Focal patches of cells with oxidative stress were observed
(arrows) increased over time in the PFP-treated lungs.

We have previously shown that gene expression of both non-enzymatic and enzymatic
antioxidants, especially those that utilize glutathione, are differentially altered
24 hours after PFP exposure depending on the post-exposure time and age [21]. We hypothesized that exposure to PFP activates the oxidative stress sensitive transcription
factor Nrf2 and upregulates phase II detoxification genes containing the antioxidant
response element (ARE). To test this hypothesis, we measured Nrf2 activation with
an in vitro luciferase reporter assay. As shown in Figure 3A, Nrf2 activation increased in a dose-dependent manner with PFP treatment. Even at
the lowest dose of 2 μg/cm2, Nrf2 activity was significantly upregulated 2-fold above controls (p<0.05). At 10 μg/cm2, Nrf2 activity was upregulated almost 6-fold above controls (p<0.01), at levels comparable to 10 μM of tert-butylhydroquinone (t-BHQ), a known Nrf2
inducer. No cytotoxic effects were observed at the highest concentration of 10 μg/cm2 PFP (data not shown).

EMSA studies (Figure 3B) confirmed that the increased Nrf2 activation is associated with an increased binding
activity of the ARE consensus element which regulates the expression of HMOX1 and
NQO1 for instance [27]. Next, we measured mRNA levels of an oxidative stress marker, HMOX1. The promoter
region of HMOX1 contains ARE and has been shown to be Nrf2 inducible, and well correlated
with both DTT consumption and PAH content in ultrafine PM [14,16]. We treated cells at 2 μg/cm2, the lowest dose from our dose-dependent data and observed a nearly 6-fold HMOX1
induction (p<0.01) after 4 hours of treatment. Treatment with Nrf2 activator t-BHQ increases HMOX1
levels; nearly 15 fold greater than controls (Figure 3C).

PFP variably upregulates antioxidants in vivo

To measure antioxidant enzymes related to these in vitro studies, we used an in vivo
exposure followed by an acute time course. We measured total antioxidant capacity
in the lung tissue, along with mRNA and protein expression of 3 categories of antioxidants:
oxidative stress (HMOX1), superoxide metabolism (SOD1, SOD2), and hydrogen peroxide
sensors (CAT, PRDX6).

First, we measured the total antioxidant capacity (TAC) of whole lung homogenate of
adult and neonates reared in either filtered air (FA) or exposed to PFPs and allowed
to recover for 2, 24 and 48 hours post exposure (PFP2, 24, 48). TAC measures were
similar among neonates and adults, and no age or exposure effects were observed (Table 3). This was a surprising result, so we estimated airway and alveolar deposition following
PFP exposure, and calculated deposition fractions of 0.195 and 0.15 for the adult
and neonatal lung, respectively, agreeing with published deposition of 0.2 [28]. Using average body weights of 16g for neonates and 300g for adults, minute ventilation
(MV) were calculated to be 25 ml/min in neonates, and 384 ml/min in adults. The estimated
deposited dose (DD) was determined, where DDneonate = 1.89 ng/g and DDadult = 2.01 ng/g. Since the DD were similar between the two ages, we used lung compartment
specific approaches and analyzed selected antioxidant enzymes. To determine whether
exposure causes oxidative stress, we measured a typical marker, HMOX1. As shown in
Figure 4, basal HMOX1 mRNA levels were comparable in between lung compartments in neonates.
Conversely, HMOX1 was maximally expressed in the adult parenchyma while HMOX1 airway
expression was similar to neonates. Post PFP exposure, we observed transient upregulation
in the neonatal parenchyma; HMOX1 mRNA were significantly upregulated between PFP2
(p<0.01) and PFP24 (p<0.05) groups. Conversely, adult HMOX1 expression was largely unchanged. HMOX1 protein
quantification revealed no exposure effects in neonates. On the contrary, adult HMOX1
was significantly upregulated at PFP48 (p<0.05). Spatial localization of HMOX1 revealed higher basal HMOX1 levels in adults.
Post PFP exposure, neonatal HMOX1 protein was present diffusely in the parenchyma
and densely localized in patches of bronchiolar epithelium. Contrastingly, adult HMOX1
protein was abundant in both the parenchyma and the bronchiolar epithelium.

Figure 4.HMOX1 mRNA and protein expression. Animals were whole body exposed to FA or PFP for 6 hours, and sampled at 2, 24 and
48 hours post exposure; denoted as PFP2, PFP24 and PFP48. RT-PCR expression in airway
and parenchymal compartments in neonatal and adult rats exposed to PFPs. (A) HMOX1 expression between compartments was similar in neonatal FA controls, but was
greatest in adult parenchyma compared to both adult airways and neonatal parenchyma.
(B) After PFP exposure, HMOX was transiently upregulated at PFP2 and PFP24 in the neonatal
parenchyma. (C) Compared to neonates, adults had a delayed upregulated HMOX1 expression in the parenchyma
that persisted to PFP48. Data are presented as mean+SEM (n=5-7 rats/group, in each
compartment), * significantly different compared to neonates in the same compartment,
† significantly different compared to airways in the same age, ‡ significantly different
compared to FA in the same compartment. (D) Representative HMOX1 Western blot with actin loading control. (E) HMOX1 blots were quantified and revealed no exposure dependent effects in the neonatal
lung. (F) HMOX1 protein expression trended upwards at 24 hours and reached statistical significance
at PFP48. Data is presented as mean+SEM (n=6 rats/group) ‡ significantly different
compared to FA in the same age. HMOX1 immunohistochemical detection of protein expression
(n=6 rats/group) is presented in neonatal (G-H) and adult (I-J) of FA controls (G, I) and PFP48 (H and J) groups. HMOX1 protein was significantly enhanced in neonatal airways in the PFP48
group compared to FA. Additionally, robustly expressed HMOX1 protein was increased
in both adult airways and parenchyma at PFP48 exposure. Scale bars are 50 μm.

PFPs alter specific antioxidant enzymes in an age and time-dependent manner

After establishing the incidence of oxidant stress, we examined the production of
superoxide, the initial ROS species formed in the sequential reduction of molecular
oxygen (Figure 1A). There are two main enzymes that perform cellular superoxide metabolism: cytosolic
copper-zinc superoxide dismutase (SOD1) and mitochondrial manganese superoxide dismutase
(SOD2). We measured mRNA and protein of both SOD1 (Figure 5) and SOD2 (Figure 6) in lung tissue. Basal cytosolic SOD1 mRNA levels were similar between lung (parenchyma
and airway) compartments and did not change with age. Conversely, SOD2 mRNA were significantly
greater in adults in both lung compartments. After PFP exposure, no discernible differences
were observed in the neonatal lung in either superoxide dismutase isoform. However,
SOD1 mRNA was significantly elevated in the adult airways at PFP48 (p<0.01). We did not find any exposure-related changes in SOD1 or SOD2 protein expression
in neonates, but found that SOD1 was upregulated more than 2-fold at PFP48, and that
SOD2 was approximately 1.7-fold higher at PFP2 in PFP exposed adults. Immunohistochemistry
revealed similar results; neonatal superoxide dismutase proteins remained unaffected
post-exposure, but heavy SOD1 protein expression in both adult airway and parenchymal
tissue were noted at PFP48.

Figure 6.SOD2 mRNA and protein expression. RT-PCR: (A) SOD2 mRNA expression is higher in adults, and highest in the adult parenchyma. (B) No exposure effects on SOD2 mRNA were observed in neonates. (C) Adult SOD2 mRNA was decreased in PFP48. Data are presented as mean+SEM (n=5-7 rats/group,
in each compartment), * significantly different compared to neonates in the same compartment,
† significantly different compared to airways in the same age, ‡ significantly different
compared to FA in the same compartment. Western blotting: (D) Scan of representative SOD2 and actin blots. (E) Neonatal whole lung SOD2 protein expression was unchanged with exposure, and (F) adult whole lung SOD2 protein trended upwards at PFP2, but was statistically insignificant.
(G-J) Immunohistochemical localization of SOD2 in lung (n=6 rats/group). SOD2 protein was
more abundant in adults compared to neonates, but no exposure specific differences
were observed. Scale bar is 50 μm.

Finally, we measured the levels of two antioxidant enzymes responsible for the breakdown
of hydrogen peroxide (HOOH) - catalase (CAT) and peroxiredoxin VI (PRDX6). As with
the previous enzymes, we quantified mRNA and protein and used immunohistochemistry
to visualize these two antioxidant enzymes within the lung. Figure 7 shows results from the studies of CAT, which catalyzes the dismutation of 2 molecules
of HOOH into H2O and molecular oxygen. Constitutive CAT levels revealed that adults have the highest
expression levels, especially in the parenchyma. Although we were unable to observe
exposure effects on neonatal CAT, a time-dependent decrease was seen in adult CAT
mRNA in PFP24 (p<0.05) group. Protein quantification mirrored the lack of response in neonates, with
CAT protein levels remaining steady post exposure. Interestingly, we observed a transient
but insignificant upregulation in adult CAT levels in the PFP2 group. CAT protein
levels reverted to FA levels at PFP24 and PFP48, corresponding with decreases seen
in mRNA. CAT immunohistochemistry revealed higher expression in the adults. Neonatal
CAT immunostaining was supportive of the protein quantification; we did not observe
any changes post exposure. Similarly, adult CAT protein was abundant in both the airways
and the parenchyma at PFP2, but reverted back to FA levels at subsequent recovery
times (data not shown). Finally, we assessed mRNA and protein expression of PRDX6
(Figure 8). PRDX6 is a highly conserved peroxidase present most abundantly in the lung [29]. Basal PRDX6 mRNA levels were consistent across age and lung compartments. Post exposure,
we observed a significant increase in neonatal parenchymal mRNA for PRDX6 (p<0.01) that was not replicated in adults. However, protein levels were inconsistent
with mRNA data, and no statistically significant differences were seen in either neonates
or adults. Immunohistochemical detection of PRDX6 protein revealed highly abundant
levels in the bronchiolar epithelium in both ages, but similar to the protein quantification
results, we did not observe any exposure-related differences in expression for either
age. A summary of PFP induced changes in antioxidant expression is compared (Table 4).

Figure 7.CAT mRNA and protein expression. RT-PCR: (A) CAT mRNA was most abundant in the adult
parenchyma, compared to neonates and the airway compartment. (B) Neonatal CAT mRNA is unaffected by PFP exposure. (C) Adult CAT expression was significantly decreased at PFP24 and PFP48. Data are presented
as mean+SEM (n=5-7 rats/group, in each compartment), * significantly different compared
to neonates in the same compartment, † significantly different compared to airways
in the same age, ‡ significantly different compared to FA in the same compartment.
(D) Representatively CAT and actin Western blots. (E) Neonatal CAT protein was unchanged post exposure. (F) Adult CAT protein expression trended upwards transiently 2 hours post exposure, but
this interaction was insignificant. (G-J) Immunohistochemical localization of CAT protein (n=6 rats/group) in lung tissue:
Under FA conditions, adults had more abundant CAT protein than neonates. Similar to
western blotting, intense protein localization to both airway and parenchyma tissue
was observed. Scale bar is 50 μm.

Discussion

We accomplished three goals in this study. First, we determined the extent of ROS
generation by PFPs. Second, we examined whether ROS in PFPs were sufficient to cause
oxidative stress and activate the Nrf2 antioxidant response pathway using an ex vivo airway epithelium model and in an in vitro reporter system. Third, we compared antioxidant responses by measuring expression
of three antioxidant enzyme families in neonatal and young adult rat lungs. We generated
PFP as a surrogate for diluted vehicular exhaust. Animals were whole-body chamber
exposed to 22.4 ± 5.6 μg/m3 PFPs, where particle number was measured to be 9.4 ± 0.5 × 104 particles/cm3, consistent with ultrafine particle values reported 30 meters downwind from a major
Los Angeles, CA highway (approx. 5.0 × 104 particles/cm3; 65nm particles) [30]. The PFP environment is rich is organic compounds, with 2-methylnaphthalene (35.9
ng/m3) and naphthalene (15.4 ng/m3) as the most abundant PAH present in the vapor and particle phases, respectively
[21]. Gasoline and diesel fuels have high concentrations of naphthalene, at 2600 mg/L
and 1600 mg/L, respectively [31]. While a portion of PAHs in diesel exhaust emission is formed during fuel combustion,
the majority of PAH are thought to be fuel-derived PAHs that are not destroyed during
combustion. Measurements of vehicle exhaust from catalyst-equipped gasoline-powered
motor vehicles emit approximately 1000 μg/km emissions of 2-methylnaphthalene and
naphthalene, respectively [32]. Many PAHs present in PM have been shown to be capable of redox cycling and are carcinogenic
[33-35]. The current study shows that, at environmentally relevant levels, PFP elicits significant
oxidant effects, capable of activating Nrf2 and antioxidant enzymes.

PFPs are potent ROS generators that are capable of generating lung oxidative stress
and activating the Nrf2-ARE pathway using in vitro and ex vivo models. We chose the DTT, HOOH and ·OH assays to measure the ability of PM to produce
ROS through reduction of molecular oxygen. Both DTT loss and HOOH production can be
from transition metals and quinones, while ·OH production typically requires transition
metals [14,25]. In the current study, the PFP DTT response of 0.56 nmol/hr/μg PM is well within
the typical range of ambient PM reported in the literature [36]. PFPs are rich in PAHs [21], which can be converted to redox-active quinones in vivo. Further, molecular oxygen is a viable electron acceptor that generates ROS (e.g.
superoxide, hydrogen and hydroxyl radicals), both through cellular respiration and
production by PM [24,25]. We hypothesized that through these mechanisms, PAHs indirectly play the primary
role in ROS generation. Transition metals are another major source of ROS from PM
[37,38], but we expected low concentrations of metals in our PM samples. Measurements of
8 common transition metals from PFPs found only zinc (Zn) above the detection limit.
Zinc cannot redox cycle in a cell-free system and is not active in either the DTT
or ·OH assay, so it is unlikely to produce any of the chemically generated ROS observed
here [36,39]. Thus, organic species in the PFP are likely responsible for DTT loss and HOOH generation
in our chemical assays.

ROS from PM exposures have been implicated in causing oxidative stress [13,40]. To visualize and detect oxidative stress in the 3-dimensional lung environment,
we used a cell-permeable probe (CellROX) that fluoresces upon oxidation by ROS in
a novel ex vivo model. We unequivocally showed that PFPs cause oxidative stress in a proximal to
distal manner in the bronchiolar epithelium; the same direction that particles were
instilled. Furthermore, as seen in the supplemental video, PFP treatment causes minor
sloughing of CellROX positive cells in the bronchiolar epithelium.

We used the human macrophage cell line (U937) as an in vitro model to measure Nrf2 activation upon PFP exposure. Macrophages are one of the major
alveolar cell types in the alveolar wall that play a critical role in homeostasis,
host defense and response to foreign substances. Further, alveolar macrophages are
responsive to organic and inorganic pollutants, such as diesel particulate and urban
dust stimuli [41], promoting the release of proinflammatory cytokines and chemokines and increased
activity of antioxidant enzymes. Nrf2-ARE is a key cellular defense pathway against
ROS and oxidative stress. Under normal conditions, Nrf2 is cytosolic and constitutively
bound to repressor Keap1. Nrf2 has a short half-life of ~15 minutes and is rapidly
degraded by ubiquitinated proteosomic degradation [42]. ROS disrupts Keap1-Nrf2 binding, where Nrf2 is subsequently translocated to the
nucleus, is phosphorylated and complexes with small Maf proteins to induce transcription
of ARE-responsive phase II detoxification genes [43,44]. The Nrf2-luciferase reporter assay and EMSA demonstrates a clear dose-dependent
increase in Nrf2 activity and increased ARE binding activity after PFP treatment.
We confirmed Nrf2 induction through mRNA induction of a typical ROS and oxidative
stress responsive ARE-containing enzyme, HMOX1. With these data, we unequivocally
showed that PFP treatment is capable of activating Nrf2-ARE in vitro.

To verify these results in an in vivo system, 7-day old neonates and young adult rats were exposed to 6 hours of PFPs,
and lung tissue was assessed at 2, 24 and 48 post exposure (designated PFP2, PFP24,
and PFP48). Compared with humans, the neonatal rats used in this study are equivalent
to newborn infants, while 8 week old rats are similar to teenagers [45]. This acute exposure tests the two extremes of a critical developmental and growth
window for the lung. A limitation of our study is the increased number of confounding
factors, especially prevalent in developmental studies. In the in vivo inhalation studies, neonatal animals were exposed to PFPs along with the dams to
reduce weaning stress, along with providing a source of warmth and nutrition. Since
neonatal animals are known to group together closely with their dam, maternal filtering
may affect the inhaled PFP dose. Furthermore, neonatal proximity with the dam and
differential feeding patterns also increases the variation seen between animals. These
factors may have an undetermined effect on the deposited dose, and could possibly
affect deposited dose estimations. Due to the heterogeneous nature of the lung, and
the fact that distinct cell populations exist in separate lung compartments that may
respond to PFP differently, we microdissected the lung tissues when practicable and
used site-specific approaches to measure expression patterns of antioxidant enzymes.
We first measured HMOX1, which is pervasive in the lung [14]. After a single PFP exposure, we saw significant HMOX1 parenchymal mRNA upregulation
in both neonates and adults. Surprisingly, and unlike adult animals, mRNA upregulation
did not translate to quantifiable HMOX1 increases in the whole lung homogenate nor
in the parenchyma. The failure of neonates to sufficiently upregulate HMOX1 in the
parenchyma has multiple implications for the developing lung. It is estimated that
approximately 50 million alveoli are present at birth, which geometrically divides
to over 300 million upon reaching adulthood [46]. Unnecessary cell turnover may disrupt alveolar maturation and investigators have
previously found that mice lacking HMOX1 have reduced surfactant protein-B expression
and are more susceptible to endotoxin [47]. The lack of HMOX1 upregulation may, in part, explain the enhanced parenchymal cytotoxicity
[21] we see in neonatal rats exposed to PFP.

Superoxide dismutases are a family of enzymes that reduce O2- anions into HOOH. There are two main intracellular isoforms; cytosolic copper-zinc
superoxide dismutase (SOD1) and mitochondrial superoxide dismutase (SOD2). Only SOD1
was upregulated after PFP exposure, suggesting that O2- is present in the cytosol. Our results are in agreement with Laing et al.,[48], showing significant SOD1 but not SOD2 upregulation in mice exposed to concentrated
ambient particles in Ohio. Furthermore, we found substantial SOD1 upregulation in
the adult parenchyma after exposure, consistent with literature indicating that alveolar
type II cells are more protected from oxidative stress due to higher expression of
SOD1 and SOD2 [49]. Our results differ from previous animal models where oxidative stress was induced
using hyperoxia and SOD1 upregulation was observed in both neonates and adults [50]. This lack of concordance suggests that hyperoxia and PM exposures may increase SOD1
expression by different mechanisms, especially in neonates. Although SOD has been
shown to be present in 7-day old neonates [51], neither SOD isoform was upregulated in the neonate animals exposed to PFP. This
reinforces our hypothesis that neonatal animals are uniquely susceptible to PFP due
to their inability to respond and upregulate antioxidant enzymes, possibly due to
overriding developmental programming that is in play during this period of active
lung growth or because of the differentiation state of the affected cell populations.

CAT and PRDX6 are intracellular peroxidases present in peroxisomes and cytosol, respectively.
Peroxidases enzymatically dismute 2 molecules of HOOH into water and molecular oxygen.
CAT has high catalytic activity that is rate-limited only by the concentration of
HOOH. Oxidative DNA damage has been shown to cause cell cytotoxicity, and ROS from
PM2.5 has been implicated to cause DNA damage [33,52]. In vitro treatment with PM2.5 supplemented with exogenous CAT or SOD ameliorated oxidative DNA damage [53]. We clearly saw enhanced CAT immunostaining in adult animals in both lung compartments
2 hours post exposure, but neonatal CAT expression appeared to be less abundant than
adults and expression was unchanged by PFP exposure. The lower expression and lack
of CAT upregulation may have implications on neonatal susceptibility to air pollution
as functional CAT is thought to have a protective role. Mice lacking CAT develop normally,
but they remove HOOH at a reduced rate and are more susceptible to oxidative stress
[54]. Epidemiological studies have found that a functional single nucleotide polymorphism
in the promoter region of CAT, G-330A that results in higher blood CAT levels, is
associated with lower risk of respiratory-related school absences in children [55] and a decreased risk of developing asthma [56].

PRDX6 is capable of reducing both hydrogen peroxide and phospholipid peroxides. However,
it is dependent on the formation of glutathione S-transferase P (GSTP1) and PRDX6
heterodimer in the presence of glutathione (GSH) to generate peroxidase activity [57]. Although we saw increases in PRDX6 mRNA levels in neonatal parenchyma at 48 hours
after PFP exposure, this was not translated into increased protein. However, it may
not be surprising that we didn’t see any induction of PRDX6, because it may be unnecessary.
PRDX6 has a turnover rate of over 5 μmol/min/mg protein [57], vastly exceeding the 0.07 nmol/hr/μg PM HOOH present in PFP. Alternatively, PRDX6
induction could be limited by GSH or possibly GSTP1. As we have previously shown,
PFP exposure significantly diminishes the amount of reduced GSH within both the airways
and parenchyma in neonates, but not in adults [22]. Additionally, unlike adults, neonates did not upregulate GSTP1 expression in response
to PFP exposure. The inability to regulate either mRNA and/or protein expression of
both PRDX6 and GSTP1, in combination with diminished glutathione in key target regions
may play a role in neonatal susceptibility to PFPs.

In summary, the present study establishes that laboratory generated PFP is a potent
generator of ROS. We further determined that PFP induces oxidative stress in both
an ex vivo and in vivo models. Because our PM is low in metals, we infer that it is likely that the organic
content of PM in an exhaust atmosphere is an important contributor to pulmonary susceptibility,
particularly in the developing lung. Antioxidant enzyme expression was variably altered
after PFP exposure, depending on age, lung compartment, and post-exposure recovery
time underscoring the importance of site specific approaches to analyze biological
effects of inhaled compounds. Cell populations are location-specific and have unique
functions within the lung. The inability of neonates to upregulate antioxidants in
the parenchyma provides a mechanism for the enhanced parenchymal toxicity to PFP we’ve
previously reported [21-23]. From the data presented, we conclude that the HMOX1 and SOD1 mRNA and protein upregulation
are suitable oxidative stress detection markers to measure effects of PFP exposure.
Moreover, we posit that the enhanced susceptibility of neonatal mice to inhaled PFPs
might be due to their inability to upregulate key oxidative stress response and antioxidant
proteins that are needed to return to cellular homeostasis.

Methods

Particle generation

Premixed flame particle (PFP) aerosols were generated using a coannular premixed flame
burner as detailed previously [20,21]. Briefly, a Pyrex-tube enclosed burner was fed a metered mixture of ethylene (212.4
cc/min), oxygen (289.2 cc/min) and argon (1499 cc/min) to generate the flame. A small
flow of oxygen (52 cc/min) flowed through the outer annulus to stabilize the flame.
Dry filtered air (FA) was added to the flow downstream and burner effluent passed
through a heated 3-way catalyst to remove NOx and CO. Finally, PFPs were diluted and mixed with clean HEPA and CBR (chemical/biological/radiological)
FA prior to entering the inhalation exposure chamber. Chambers were maintained at
−0.3 inches of H2O gauge pressure and temperature were maintained between 22.2 and 24.4°C. PFP are
on average 70.6 ± 1.5 nm (geometric mean ± SD) as determined by Scanning Mobility
Particle Sizer (SMPS) measurements. Particle mass concentration in the chamber was
22.4 ± 5.6 μg/m3 PFP (mean ± SD), and the total particle numbers was (9.37 ± 0.48) ×104 (mean ± SD) determined used a condensation particle counter. Particles were high
in organic carbon and had an EC:OC ratio of 0.58. Gas- and particle-phase concentrations
of polycyclic aromatic hydrocarbons (PAHs) were 405 and 54 ng/m3, respectively.

Ambient PM1.8 collection

Ambient PM1.8 was collected in Fresno, CA during the summer of 2008 from 10:00 am to 4:00 pm local
time for two consecutive five-day periods with a two-day break, (August 24th – 28th and August 31st – September 4th 2008). PM1.8 were collected onto Teflon filters using a high volume sampler with a PM1.8 impaction stage. Additional PM collection details can be found in [38]. After collection, samples were stored at −20°C in the dark.

PM incubation

A filter section with known PM mass (18.4 μg for PFP and 115.7 μg for ambient PM1.8) was incubated in the reaction mixture for soluble metals, DTT, ·OH and HOOH analysis
along with 18.4 mM of trifluoroethanol as a filter wetting agent. Samples were stirred
constantly on a shake table to extract PM. A blank filter of the same size was also
analyzed and used to filter-blank correct all results.

Hydrogen peroxide assay

The rate of formation of HOOH was quantified from PM in 4.0 mL SLF using the method
previously described [38], except that we use the four antioxidants stated above instead of only ascorbate.
Briefly, HOOH production from PM was measured in triplicate at 0, 0.5, 1 and 1.5 hours,
and the rate of HOOH production was calculated from the slope of the linear response.
The rate of HOOH production from the filter blank was subtracted from the rate of
HOOH from PM. HOOH was quantified using HPLC with post-column derivatization and fluorescence
detection (excitation 320 nm, emission 400 nm) [58]. Daily quality controls include a solution blank and positive control (250 nM copper)
run with each experiment. HOOH calibrations were run daily, and the concentration
of the HOOH stock was confirmed using UV–VIS absorption at 240 nm.

Hydroxyl radical assay

The rate of ·OH formation from PM was quantified in 6.0 mL SLF with four antioxidants
(see above) [39]. Briefly, ·OH was quantitatively trapped using a sodium benzoate probe. The stable
product, p-hydroxybenzoic acid, was quantified using HPLC with UV–VIS detection using
a standard p-hydroxybenzoic acid solid as a daily calibration. ·OH was measured in
triplicate at 0, 1, 2, 4 and 24 hours and the rate of ·OH production was calculated
from the slope of the linear data. The rate of ·OH production from a filter blank
was subtracted from the rate of ·OH production from PM. A solution blank and a positive
control (1.44 μM iron) were run daily for quality control.

DTT assay

The DTT assay is a cell-free, in-vitro, chemical measure of the oxidative potential of PM which responds to trace redox-active
chemicals in PM such as quinones and transition metals, and is described in detail
in the literature [24,36]. Briefly, the loss of 100 μM of DTT (Arcos Organics) incubated with PM in 3.0 mL
of 0.1 M phosphate buffer (pH 7.3-7.4) at 37°C is measured over time. The rate of
DTT loss provides a quantitative measure of the oxidative potential of the PM under
conditions of the DTT assay.

Soluble metals analysis

A filter section (see above) was added to 0.50 mL of HOOH SLF and the solution was
mixed on a shake table for 24 hours at room temperature. 0.40 mL of solution was filtered
through a 0.22 μM PTFE filter (Millipore) into 3.6 mL of 3% nitric acid (trace metal
grade). Soluble metals were quantified using an inductively coupled plasma mass spectrometer
(ICP-MS) by the UC Davis interdisciplinary center for ICP-MS. A filter blank was also
analyzed and the limit of quantification was calculated as 10 time the standard deviation
of the blank [59].

Cell culture and transient transfection

Human U937 monocytic cells were obtained from the American Tissue Culture Collection
(Manassas, VA) and maintained in RPMI 1640 medium containing 10% fetal bovine serum
(Gemini, Woodland, CA), 100 U penicillin, and 100 μg/ml streptomycin supplemented
with 4.5 g/L glucose, and 1 mM sodium pyruvate. Cell culture was maintained at a cell
concentration between 2 × 105 and 2 × 106 cells/ml. For differentiation into macrophages, U937 cells were treated with TPA
(3 μg/ml) and allowed to adhere for 48 hr in a 5% CO2 tissue culture incubator at 37°C, after which they were fed with TPA-free medium.
Differentiation state was assessed by attached cell phenotype and increased expression
of MAC-2. U937 macrophages were treated in regular growth medium containing 10% FBS.
PFPs were used at 2, 5 or 10 μg/cm2 growth area, equivalent to 10, 25 and 50 μg/mL. Concentrations of PFP are preferentially
expressed in micrograms per square centimeter growth area because particles rapidly
sediment onto the cell layer.

Cell viability assay

To assess the effect of PFP on viability of U937 macrophages, we used the trypan blue
exclusion test. A 10 μl sample of re-suspended cell pellet was placed in 190 μl PBS
with 200 μl trypan blue (0.5% dilution in 0.85% NaCl). After 5 minutes, 10 μl of the
cell suspension was loaded into a hemocytometer and determined the proportion of nonviable
to viable cells.

In vitro RNA isolation and real time reverse transcription-PCR

Total RNA was isolated from U937 cells using a Quick-RNA Mini prep isolation kit (Zymo
Research, Irvine, CA), and cDNA synthesis was done as previously described [61]. Quantitative detection of heme oxygenase-1 (HMOX1, NCBI RefSeq: NM_010442.2) and
Rps13 (NCBI RefSeq: NM_026533.3) genes was performed with a StepOnePlus™ Real-Time
PCR System (Applied Biosystems) using the Fast SYBR Green Master Mix (Life Technologies,
Grand Island, NY) according to the manufacturer’s instructions. DNA-free total RNA
(1.0 μg) was reverse-transcribed using 4 U Omniscript reverse transcriptase (RT; Qiagen)
and 1 μg oligo(dT)15 in a final volume of 40 μl. The primers for each gene were designed on the basis
of the respective cDNA or mRNA sequences using OLIGO primer analysis software provided
by Steve Rozen and the Whitehead Institute/MIT Center for Genome Research [62] so that the targets were 100–200 bp in length. Primers for human HMOX1 are: left
primer ‘cacgcatatacccgctacct’ and right primer ‘ccagagtgttcattcgagca.’ Primers for
human Rps13 are: left primer ‘gtccgaaagcaccttgagag’ and right primer ‘agcagaggctgtggatgact.’
PCR amplification was carried out in a total volume of 20 μl containing 2 μl cDNA,
10 μl 2 × Fast SYBR Green Master Mix, and 0.2 μM of each primer. The PCR cycling conditions
were 95°C for 30 sec followed by 40 cycles of 94°C for 3 sec, and 60°C for 30 sec.
Detection of the fluorescent product was performed at the end of the 72°C extension
period. Negative controls were concomitantly run to confirm that the samples were
not cross-contaminated. A sample with DNase- and RNase-free water instead of RNA was
concomitantly examined for each of the reaction units described above. To confirm
the amplification specificity, the PCR products were subjected to melting curve analysis.

Ex-vivo based oxidative stress detection

Eight-week old male Sprague Dawley rats (Harlan Laboratories, Hayward, CA) were acclimated
in CBR filtered air (FA) for a week prior to experimentation. Rats were provided with
Laboratory Rodent Diet (Purina Mills, St. Louis, MO) and water ad libitum. Animals were euthanized by intraperitoneal (IP) injection of an overdose of pentobarbital
(150 mg/kg). All animal experiments were performed as described in protocols approved
by the University of California IACUC in accordance with guidelines set by the National
Institute of Health. At necropsy, tracheas were cannulated, thorax opened and lung
removed en bloc, inflated with low gelling temperature and processed as previously
described [63]. The right cranial lobe was glued to a 22 mm2 coverslip (Corning Life Sciences, Lowell, MA) with Nexaband tissue adhesive (Abbott
Animal Health, North Chicago, IL) and bisected in half to expose the main axial airway.
Agarose was removed from the bronchiolar airways. Airways were directly treated with
either 10 μg of PFP, dissolved in endotoxin free PBS (Enzo Life Sciences, Farmingdale,
NY) at 5 μg/μl concentration or PBS only (vehicle control) by pipetting the substance
onto the airway lumen in proximal to distal direction. The lungs were incubated in
Live Cell Imaging Solution (Life Technologies, Grand Island, NY) at 37°C for 30 minutes.
Then, 5 μM CellROX Deep Red Reagent (Life Technologies, Grand Island, NY) was applied
directly onto the dissected lung and allowed to incubate for 30 minutes following
manufacturer’s instructions. Lungs were washed 3 times with Live Cell Imaging Solution
and imaged continuously on the Leica TCS LSI zoom confocal microscopes (Leica Microsystems,
Wetzlar, Germany) using a 488 nm excitation laser. Experiments were repeated 5 times
on separate days with different animals.

Animal exposure protocol

Eight-week old male adult and newborn male Sprague Dawley rats with accompanying dams
(Harlan Laboratories, Hayward, CA) were allowed to acclimate in FA until newborns
reached 7 postnatal days of age in a 12 hour light/dark cycle starting at 7AM. Adult
rats were housed in steel wire cages, while newborn rats and accompanying dams were
housed in polycarbonate cages with wire tops and provided with Laboratory Rodent Diet
(Purina Mills, St. Louis, MO) and water ad libitum. Six animals per group were exposed to 22.4 ± 5.6 μg/m3 PFP or FA atmosphere for 6 hours in two separate chambers as previously described
[20,21]. Chambers are maintained at 22-23°C and 40-44% humidity. Animals were necropsied
at 2, 24 and 48 hours following the single 6-hour exposure; treated groups will be
designated as PFP2, PFP24 and PFP48, respectively. All animals were euthanized by
an intraperitoneal injection of 0.5 ml/kg body weight pentobarbital and subsequently
exanguinated prior to lung removal. At necropsy, the trachea was cannulated, thorax
opened and lung removed en bloc for processing.

In vivo lung compartmental RNA isolation and RT-PCR

Lung compartmental RNA was isolated from microdissected intrapulmonary airways and
surrounding parenchymal tissue from RNAlater (Ambion, Austin, TX) stabilized lung
tissue using the Qiagen RNeasy Mini Kit (Qiagen, Valencia, CA) as previously described
[65]. RNA purity was confirmed using spectrophotometric absorbance at 260/280 nm. Gene
quantification in the airway and parenchymal compartments were performed using inventoried
Taqman probes and primers (Applied Biosystems, Foster City, CA) listed in Table 5 as previously described [65,66]. Results were calculated using the comparative Ct method [67,68] using Hypoxanthine-guanine phosphoribosyltransferase (HPRT) as the reference gene.
HPRT was chosen as the reference gene due to consistency by age and low variance between
exposure groups as previously assessed [21,69]. Results are expressed as a fold change in gene expression relative to filtered air
exposed animals of the same age, unless otherwise stated.

Immunohistochemistry

Lungs were inflated with 37% formaldehyde vapor bubbled under 30 cm hydrostatic pressure
for 1 hour as previously described [70,71]. Samples were stored in 1% paraformaldehyde for less than 24 hours prior to tissue
processing and paraffin embedment. Paraffin sections on poly-L-lysine coated slides
from all groups were stained simultaneously to minimized variability between runs
and were immunostained for rabbit anti-SOD1 (Abcam, Cambridge, MA) at 1:3000, rabbit
anti-HMOX1 (Abcam) at 1:250, rabbit anti-PRDX6 (LabFrontier, Seoul, Korea) at 1:3000,
sheep anti-SOD2 (The Binding Site, San Diego, CA) at 1:2000 and sheep anti-CAT (The
Binding Site) at 1:2000. The concentration of primary antibody was determined through
a series of dilutions to optimize for staining density while minimizing background.
This procedure was performed according to previously described methods [63,69] with several alterations. Following tissue hydration, endogenous peroxidase activity
was quenched with a 10% solution of hydrogen peroxide. To eliminate nonspecific primary
antibody binding, tissue sections were blocked with 5% albumin. Primary antibodies
were allowed to incubate in a humidified chamber overnight at 4°C. Signal was amplified
using the Vectastain IgG Avidin-Biotin-HRP Kit (Vector Labs, Burlingame, CA), and
visualized using nickel chloride enhanced 3,3’-diaminobenzidine tetrachloride (Sigma-Aldrich,
St. Louis, MO) as the chromagen. Controls included substitution of primary antibody
with phosphate-buffered saline to ensure specific positive staining.

Statistics

Data are reported as mean ± standard error of the mean (SEM) unless otherwise stated.
Multivariate analysis of variance (MANOVA) was applied against age, compartment and
exposure factors when appropriate. Pair-wise comparisons were performed individually
using a one-way ANOVA followed by Dunnett’s post hoc tests against age and compartment
controls using StatView (SAS, Cary, NC). P values of < 0.05 were considered statistically significant.

Competing interests

One Author, Dr. Laura Van Winkle, has identified a potential apparent competing financial
interest with the American Petroleum Institute (API). Dr. Van Winkle has received
research grants from API to study the kinetics of naphthalene bioactivation and cytotoxicity
in the respiratory system and has received honoraria from API for speaking at research
conferences sponsored by API on naphthalene. API did not fund the work presented in
the attached study and the research grant funded by API has complete freedom to publish
the results regardless of whether they are in API’s interest, and without input from
API, in keeping with University of California policy. The remaining authors declare
they have no actual or potential competing financial interests.

Acknowledgments

Support for the University of California at Davis core facilities used in this work:
the Cellular and Molecular Imaging Core Facility, the confocal microscope (S10RR-026422)
and the inhalation exposure facility at the California National Primate Research Center
(RR00169). Although the research described in the article has been funded primarily
by the United States Environmental Protection Agency (EPA) through grant RD-83241401-0
to the University of California, Davis, it has not been subject to the Agency’s required
peer and policy review and, therefore, does not necessarily reflect the views of the
Agency and no official endorsement should be inferred. The views expressed in this
publication are solely those of the authors, and EPA does not endorse any products
or commercial services mentioned in this publication. Miss Charrier’s effort was supported
by STAR Fellowship Assistance Agreement no. FP-91718101-0 awarded by the U.S. EPA.
The project was also supported in part from the National Institute of Environmental
Health Sciences (P42ES004699 and R01 ES019898). Dr. Chan’s effort was supported by
a training program in Environmental Health Sciences (T32ES007059). The content is
solely the responsibility of the authors and does not necessarily represent the official
views of the National Institute of Environmental Health Sciences or the National Institutes
of Health. We would also like to acknowledge Yongjing Zhao, Walter Ham, Mike Kleeman,
Chris Ruehl and Norman Kado for their expertise and aid in ambient PM collection.